Influenza vaccine, composition, and methods of use

ABSTRACT

The invention relates to compositions and vaccines that include a mutated  Bordetella  strain for treating or preventing an influenza infection in a mammal. In addition, the invention further provides methods for protecting a mammal against infection by influenza and/or eliciting an immune response against an influenza virus in a mammal using the composition or vaccine.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S. patentapplication Ser. No. 15/353,758 filed on Nov. 17, 2016, which is acontinuation application of U.S. patent application Ser. No. 13/377,070filed on Feb. 2, 2012 (now U.S. Pat. No. 9,526,778) which is a nationalstage entry application under 35 U.S.C. 371 of international patentapplication number PCT/IB09/07153 filed on Jun. 15, 2009, whichdesignated the U.S.

FIELD

The invention relates to fields of microbiology and virology.

BACKGROUND

There are three types of influenza virus: A, B, and C, which varygreatly in their epidemiological pattern. Influenza A virus is both thebest characterized and the most serious threat to public health, capableof inducing massive epidemics or pandemics. This virus is also highlyvariable antigenically, making effective vaccine production difficult.

A vaccine to influenza would be one of the most efficacious, safe,nontoxic, and economical weapons to prevent disease and to control thespread of the disease. The primary aim of vaccination is to activate theadaptive specific immune response, primarily to generate B and Tlymphocytes against antigen(s) associated with the disease or thedisease agent.

Currently some vaccines against influenza are available and primarilyconsist of inactivated vaccines. These vaccines can comprise two type Aantigens (e.g., H1N1 and H3N2) and one type B antigen. The availablevaccines typically include whole virion, split-product, and subunitvaccines. Generally, these vaccines are effective in up to 90% ofvaccinated individuals if the vaccines closely match the identity of theemerging epidemic. However, they need to be updated each year to keeppace with antigenic drift of the influenza virus.

Moreover, a cold-adapted live attenuated intranasal vaccine (LAIV)against seasonal influenza has been recently described. Cross-protectiveimmunity was demonstrated in a study which reported protection against aH3N2 virus in cotton rats infected with a H1N1 strain. However, onemajor concern linked with LAIV is the possibility of genetic reversionand re-assortment with wild-type influenza viruses, resulting in a new,potentially infectious strain.

The invention addresses these and other problems in the influenzavaccine field by providing a vaccine and composition that elicits animmune response against one or more influenza strains using a mutatedBordetella strain as the active principle agent.

In addition, the related art describes various types of vaccines andcompositions using Bordetella strains (WO2007104451 and WO2003102170) toinduce immune responses against, e.g., Bordetella bacteria capable ofcausing whooping cough in humans; however the art fails to disclosemethods or compositions for eliciting an immune response to an influenzavirus in a mammal using the methods, compositions, and/or vaccines ofthe invention.

Thus, a need exists for a novel influenza vaccine capable of providingbroad protection against diseases caused by influenza virus infection.

BRIEF SUMMARY

The invention relates to compositions and vaccines that include amutated Bordetella strain for treating or preventing an influenzainfection in a mammal. In addition, the invention further providesmethods for protecting a mammal against infection by influenza and/oreliciting an immune response against an influenza virus in a mammalusing the composition or vaccine.

In one aspect, the invention provides a method of eliciting an immuneresponse against an influenza virus in a mammal, comprising:administering a mutated Bordetella strain to the mammal, wherein thestrain comprises a mutated pertussis toxin (ptx) gene, a deleted ormutated dermonecrotic (dnt) gene, and a heterologous ampG gene. In someaspects, the Bordetella strain comprises a Bordetella pertussis strain.In some such aspects, the wild-type Bordetella strain ampG gene isreplaced by an E. coli ampG gene. In other aspects, the mutation of theptx gene comprises the substitution of an amino acid involved insubstrate binding and/or an amino acid involved in catalysis. In somesuch aspects, the substitution of the amino acid involved in substratebinding comprises R9K and the substitution of the amino acid involved incatalysis comprises E129G. In some aspects, the Bordetella straincomprises a triple mutant strain. In some such aspects, the Bordetellastrain comprises a BPZE1 strain. In other such aspects, the Bordetellastrain is attenuated. In some aspects, the Bordetella strain comprises alive strain. In other aspects, the Bordetella strain does not comprise aheterologous gene other than the heterologous ampG gene. In someaspects, the Bordetella strain does not comprise a heterologousexpression platform to carry heterologous antigens to the respiratorymucosa of the mammal. In other aspects, the methods further comprise theprevention or treatment of the influenza infection in the mammal. Insome aspects, the Bordetella strain is administered prior to theinfluenza infection. In some such aspects, the Bordetella strain isadministered about 6 weeks or more prior to the influenza infection. Inother such aspects, the Bordetella strain is administered about 12 weeksor more prior to the influenza infection. In some aspects, the influenzavirus comprises H3 or H1. In other aspects, the influenza viruscomprises N2 or N1.

In some aspects, the influenza virus comprises H3 and N2. In otheraspects, the influenza virus comprises H1 and N1. In some aspects, theimmune response comprises a Th1 immune response. In some aspects, thestrain is administered to the mammal by subcutaneous (s.c.), intradermal(i.d.), intramuscular (i.m.), intravenous (i.v.), oral, or intranasaladministration; or by injection or by inhalation. In other aspects, thestrain is administered intranasally. In some other aspects, the strainis administrated to a mammal in need of protective immunity against theinfluenza infection. In some aspects the mammal is a child. In someaspects, the strain is administered once in a single dose. In someaspects, the strain is administered in more than one dose. In some suchaspects, the strain is administered twice in two doses. In otheraspects, the two doses are administered about 3 weeks apart. In someaspects, a level of protection against the influenza infection is morethan about 60%. In other aspects, a level of protection against theinfluenza infection is more than about 50%. In some aspects, the mammalis a human.

In another aspect, the invention provides methods of eliciting aprotective immune response against an H3N2 influenza virus in a human,comprising: intranasally administering a live and attenuated BPZE1strain to the human prior to infection of the human by the H3N2influenza virus, wherein the strain does not comprise a heterologousexpression platform to carry heterologous antigens to the respiratorymucosa of the human.

In another aspect, the invention provides methods of eliciting an immuneresponse against an influenza virus in a human, comprising:administering a live Bordetella strain to the human, wherein the straindoes not comprise a heterologous expression platform to carryheterologous antigens to the respiratory mucosa of the human.

In another aspect, the invention provides methods of protecting a mammalagainst a disease caused by an influenza infection, comprising:administering to the mammal a mutated Bordetella strain comprising amutated ptx gene, a deleted or mutated dnt gene, and a heterologous ampGgene.

In another aspect, the invention provides a protective immunity againstan influenza infection, comprising: administering to the mammal amutated Bordetella strain comprising a mutated ptx gene, a deleted ormutated dnt gene, and a heterologous ampG gene.

In another aspect, the invention provides a composition for treating orpreventing an influenza infection in a mammal, comprising: a mutatedBordetella strain, wherein the strain comprises a mutated pertussistoxin (ptx) gene, a deleted or mutated dermonecrotic (dnt) gene, and aheterologous ampG gene. In some aspects, the Bordetella strain comprisesa Bordetella pertussis strain. In other aspects, the wild-typeBordetella strain ampG gene is replaced by an E. coli ampG gene. In someaspects, the mutation of the ptx gene comprises the substitution of anamino acid involved in substrate binding and/or an amino acid involvedin catalysis. In other aspects, the substitution of the amino acidinvolved in substrate binding comprises R9K and the substitution of theamino acid involved in catalysis comprises E129G. In some aspects, theBordetella strain comprises a triple mutant strain.

In another aspect, the invention provides a vaccine comprising acomposition of the invention for treating or preventing the influenzainfection in the mammal. In some aspects, the vaccine comprises aBordetella strain composition described herein. In some aspects, thevaccine is formulated for intranasal administration.

In another aspect, the invention provides a Bordetella strain identifiedby accession number CNCM 1-3585.

In another aspect, the invention provides a Bordetella strain identifiedby accession number V09/009169.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the invention willbecome better understood with regard to the following description, andaccompanying drawings, where:

FIGS. 1A and 1B show protection rate of BPZE1-treated mice againstlethal challenge with mouse-adapted H3N2 virus. Adult Balb/c mice werenasally administered with 5×10⁶ cfu of BPZE1 bacteria and challengedeither 3 weeks (solid square) or 6 weeks (solid triangle) later with alethal dose (2LD50) of mouse-adapted H3N2 virus. Body weight changeswere monitored daily and mice were euthanized when body weight lossexceeded 20% of the original body weight. Survival rates were comparedto non-treated mice (solid lozenge). 10 animals per group were assessed.Results are representative of three independent experiments.

FIG. 2 shows protection rates against lethal challenge withmouse-adapted H3N2 virus in mice treated with dead versus live BPZE1bacteria. Adult Balb/c mice were nasally administered with 5×10⁶ cfu oflive (solid triangle) or dead (solid square) BPZE1 bacteria, andchallenged 6 weeks later with a lethal dose (2LD50) of mouse-adaptedH3N2 virus. Body weight changes were monitored daily and mice wereeuthanized when body weight loss exceeded 20% of the original bodyweight. Survival rates were compared to non-treated mice (solidlozenge). 10 animals per group were assessed. Results are representativeof two independent experiments.

FIGS. 3A and 3B show protection rate against lethal challenge withmouse-adapted H3N2 virus in mice treated twice with live BPZE1 bacteria.Adult Balb/c mice were nasally administered twice at a 4-week intervalwith 5×10⁶ cfu of BPZE1 bacteria (solid square) and challenged 4 weekslater with a lethal dose (2LD50) of mouse-adapted H3N2 virus. Bodyweight changes were monitored daily and mice were euthanized when bodyweight loss exceeded 20% of the original body weight. Survival rateswere compared to non-treated mice (solid lozenge). 10 animals per groupwere assessed. Results are representative of two independentexperiments.

FIGS. 4A and 4B show protection rate of BPZE1-treated mice againstlethal challenge with H1N1 influenza A virus. Adult Balb/c mice werenasally administered three times at 4-week and 3-week intervals (solidtriangle) with 5×10⁶ cfu of live BPZE1 bacteria. The animals werechallenged with a lethal dose (4 LD50) of A/PR/8/34 (H1N1) influenza Avirus 2 weeks after the last BPZE1 treatment. Body weight changes weremonitored daily and mice were euthanized when body weight loss exceeded20% of the original body weight. Survival rates were compared tonon-treated mice (solid lozenge). 10 animals per group were assessed.Results are representative of 2 independent experiments.

FIG. 5 shows viral load quantification in the lungs of protected versusnon-protected mice. Adult Balb/c mice were nasally administered twice ata 4-week interval with 5×10⁶ cfu of live BPZE1 bacteria and challenged 4weeks later with a lethal dose (2LD50) of mouse-adapted H3N2 virus. Fiveanimals per group were sacrificed 3 days post-viral challenge, theirlungs were harvested and individually processed for in vitrodetermination of TCID₅₀ upon infection of MDCK cells. The viral load wascompared with that obtained in non-treated mice. Results arerepresentative of 3 independent experiments.

FIGS. 6A and 6B show lung histology and cellular infiltrates and CD3⁺ Tcell population in the lungs in BPZE1-treated versus non-treated miceafter lethal viral challenge. Adult Balb/c mice were nasallyadministered with 5×10⁶ cfu of live BPZE1 bacteria and challenged 6weeks later with a lethal dose (2LD50) of mouse-adapted H3N2 virus.Three days post-viral challenge, mice were euthanized and their lungswere individually processed for histology analysis (A) orbroncho-alveolar lavages for analysis of the cellular infiltrates (B).Legend A: Infected control mice displayed severe inflammation, pulmonaryedema (black arrow) and severe necrotizing bronchitis with necrotic celldebris (open arrow). Infected BPZE1-treated mice showed only minimalinflammation and airway damage, and mild peribronchular damage.Representative fields are shown. Results were equivalent in more than 40fields analyzed per group (>5 fields/section, 2 sections/mouse and 4mice/group). Legend B: a, naïve mice non-challenged; b, naïve micechallenged with H3N2 virus; c, BPZE1-treated mice non-challenged; d,BPZE1-treated mice challenged with H3N2 virus. Four animals per timepoint per group were individually assessed. **, p≤0.01; ***, p≤0.001.Results are representative of two independent experiments. (C). FACSanalysis of the CD3⁺ T cell population in the mice lungs. 3 days or 5days after lethal H3N2 influenza virus challenge, non-treated controlmice and mice treated twice with BPZE1 were euthanized and CD3⁺ T cellpopulation in their lungs were analyzed by flow cytometry. Four animalsper time point per group were individually assessed. Results areexpressed in percentage of CD3⁺ T cells in the total lung cellpopulation. mean±SD. Legend: a, naïve mice non-challenged; b,BPZE1-treated mice non-challenged; c, naïve mice challenged with H3N2virus and sacrificed 3 days later; d, BPZE1-treated mice challenged withH3N2 virus and sacrificed 3 days later; e, naïve mice challenged withH3N2 virus and sacrificed 5 days later; f, BPZE1-treated mice challengedwith H3N2 virus and sacrificed 5 days later. ***, p≤0.001.

FIGS. 7A-7L show pro- and anti-inflammatory cytokine and chemokineprofiles in the BPZE1-treated versus non-treated mice after lethal H3N2viral challenge. Adult Balb/c mice were nasally administered twice at a4-week interval with 5×10⁶ cfu of live BPZE1 bacteria and werechallenged with a lethal dose (2LD50) of mouse-adapted H3N2 virus 4weeks after the last administration. One and three days post-viralchallenge, five mice per group and per time point were sacrificed andbroncho-alveolar lavages fuilds (BALFs) were collected. 14inflammation-related cytokines and chemokines were measured in eachindividual BALF sample. Legend: 1, non-treated non-challenged mice; 2,BPZE1-treated non-challenged mice; 3, non-treated challenged mice andsacrificed 1 day post-challenge; 4, BPZE1-treated challenged mice andsacrificed 1 day post-challenge; 5, non-treated challenged mice andsacrificed 3 days post-challenge; 6, BPZE1-treated challenged mice andsacrificed 3 days post-challenge. *, p<0.05; **, p≤00.01; ***, p≤00.001.

FIGS. 8A-8C show role of the B. pertussis-specific immunity in thecross-protection. (A). Naive (open triangle) or anti-H3N2 (solid circle)or anti-BPZE1 (open circle) immune serum was ip. injected to adult naïveBalb/c mice one day prior to lethal challenge (2LD50) with mouse-adaptedH3N2 virus. Body weight changes were monitored daily and mice wereeuthanized when body weight loss exceeded 20% of the original bodyweight. Survival rates were compared to non-treated mice (solidtriangle). 10 animals per group were assessed. (B) & (C). Adult Balb/cmice were nasally administered with 5×10⁶ cfu of live BPZE1 bacteria and6 weeks later the animals were euthanized and their spleens wereharvested. Pooled (B) or individual (C) splenocytes from 6 animals werestimulated with either BPZE1 lysate or heat-inactivated H3N2 virusparticles. 3H-thymidine incorporation (B) and IFN-γ ELISPOT assay (C)were performed as described below. Representative of two differentexperiments, both showed similar results; mean±SD; *, p<0.05, ***,p<0.001.

DETAILED DESCRIPTION Introduction and Overview

The invention relates to compositions and vaccines that include amutated Bordetella strain for treating or preventing influenza infectionin a mammal. In addition, the invention further provides methods forprotecting a mammal against infection by influenza and/or eliciting animmune response against an influenza virus in a mammal using thecomposition or vaccine.

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

As used herein, the abbreviation “PTX” refers to pertussis toxin, whichsynthesizes and secretes an ADP-ribosylating toxin. PTX is comprised offive different subunits (named S1-S5) with each complex containing twocopies of S4. The subunits are arranged in an A-B structure. The Acomponent is enzymatically active and is formed from the S1 subunit,while the B component is the receptor binding portion and is made up ofsubunits S2-S5.

As used herein the abbreviation “DNT” refers to pertussis dermonecrotictoxin, which is a heat labile toxin that can induce localized lesions inmice and other laboratory animals when it is injected intradermally.

As used herein the abbreviation “TCT” refers to tracheal cytotoxin,which is a virulence factor synthesized by Bordetellae. TCT is apeptidoglycan fragment and has the ability to induce interleukin-1production and nitric oxide synthase. It has the ability to cause stasisof cilia and has lethal effects on respiratory epithelial cells.

The term “attenuated” refers to a weakened, less virulent Bordetellastrain that is capable of stimulating an immune response and creatingprotective immunity, but does not generally cause illness.

The term “rapid protective immunity” means that immunity againstBordetella is conferred in a short time after administration of themutated Bordetella strain of the invention.

The term “Bordetella strain” or “strain” includes strains fromBordetella pertussis, Bordetella parapertussis, and Bordetellabronchiseptica.

The term “child” is meant to be a person or a mammal between 0 monthsand 18 years of age.

“Treating” refers to any indicia of success in the treatment oramelioration or prevention of the disease, condition, or disorder,including any objective or subjective parameter such as abatement;remission; diminishing of symptoms or making the disease condition moretolerable to the patient; slowing in the rate of degeneration ordecline; or making the final point of degeneration less debilitating.The treatment or amelioration of symptoms can be based on objective orsubjective parameters; including the results of an examination by aphysician. Accordingly, the term “treating” includes the administrationof the compounds or agents of the invention to prevent or delay, toalleviate, or to arrest or inhibit development of the symptoms orconditions associated with a disease, condition or disorder as describedherein. The term “therapeutic effect” refers to the reduction,elimination, or prevention of the disease, symptoms of the disease, orside effects of the disease in the subject. “Treating” or “treatment”using the methods of the invention includes preventing the onset ofsymptoms in a subject that can be at increased risk of a disease ordisorder associated with a disease, condition or disorder as describedherein, but does not yet experience or exhibit symptoms, inhibiting thesymptoms of a disease or disorder (slowing or arresting itsdevelopment), providing relief from the symptoms or side effects of adisease (including palliative treatment), and relieving the symptoms ofa disease (causing regression). Treatment can be prophylactic (toprevent or delay the onset of the disease, or to prevent themanifestation of clinical or subclinical symptoms thereof) ortherapeutic suppression or alleviation of symptoms after themanifestation of the disease or condition.

“Concomitant administration” of a known drug (or other compound) withthe composition of the invention means administration of the drug (orother compound) together with the composition at such time that both theknown drug (or other compound) will have a therapeutic effect ordiagnostic effect. Such concomitant administration can involveconcurrent (i.e., at the same time), prior, or subsequent administrationof the drug (or other compound) with respect to the administration of acomposition of the invention. A person of ordinary skill in the artwould have no difficulty determining the appropriate timing, sequence,and dosages of administration for particular drugs (or other compounds)together with compositions of the invention.

The terms “protection” and “prevention” are used herein interchangeablyand mean that an infection by influenza is impeded.

“Prophylaxis vaccine” means that this vaccine prevents influenzainfection upon future exposure.

The term “immunogenic composition” or “composition” means that thecomposition can induce an immune response and is therefore antigenic. By“immune response” means any reaction by the immune system. Thesereactions include the alteration in the activity of an organism's immunesystem in response to an antigen and can involve, for example, antibodyproduction, induction of cell-mediated immunity, complement activation,or development of immunological tolerance.

As used herein, the term “disease” has the meaning generally known andunderstood in the art and comprises any abnormal condition in thefunction or well being of a host individual. A diagnosis of a particulardisease by a healthcare professional can be made by direct examinationand/or consideration of results of one or more diagnostic tests.

The terms “live vaccine composition”, “live vaccine”, “live bacterialvaccine”, and similar terms refer to a composition comprising a strainof live Bordetella bacteria that provides at least partial protectiveimmunity against influenza.

The terms “oral”, “enteral”, “enterally”, “orally”, “non-parenteral”,“non-parenterally”, and the like, refer to administration of a compoundor composition to an individual by a route or mode along the alimentarycanal. Examples of “oral” routes of administration of a compositioninclude, without limitation, swallowing liquid or solid forms of avaccine composition from the mouth, administration of a vaccinecomposition through a nasojejunal or gastrostomy tube, intraduodenaladministration of a vaccine composition, and rectal administration,e.g., using suppositories that release a live bacterial vaccine straindescribed herein.

The term “topical administration” refers to the application of apharmaceutical agent to the external surface of the skin or the mucousmembranes (including the surface membranes of the nose, lungs andmouth), such that the agent crosses the external surface of the skin ormucous membrane and enters the underlying tissues. Topicaladministration can result in a limited distribution of the agent to theskin and surrounding tissues or, when the agent is removed from thetreatment area by the bloodstream, systemic distribution of the agent.In a preferred form, the agent is delivered by transdermal delivery,e.g., using a transdermal patch. Transdermal delivery refers to thediffusion of an agent across the skin (stratum corneum and epidermis),which acts as a barrier few agents are able to penetrate. In contrast,the dermis is permeable to absorption of many solutes and drugs, andtopical administration therefor occurs more readily through skin whichis abraded or otherwise stripped of the epidermis to expose the dermis.Absorption through intact skin can be enhanced by combining the activeagent with an oily vehicle (e.g., creams, emollients, penetrationenhancers, and the like, as described, e.g., in Remington'sPharmaceutical Sciences, current edition, Gennaro et al., eds.) prior toapplication to the skin (a process known as inunction).

The term “nasal administration” refers to any form of administrationwhereby an active ingredient is propelled or otherwise introduced intothe nasal passages of a subject so that it contacts the respiratoryepithelium of the nasal cavity, from which it is absorbed into thesystemic circulation. Nasal administration can also involve contactingthe olfactory epithelium, which is located at the top of the nasalcavity between the central nasal septum and the lateral wall of eachmain nasal passage. The region of the nasal cavity immediatelysurrounding the olfactory epithelium is free of airflow. Thus,specialized methods must typically be employed to achieve significantabsorption across the olfactory epithelium.

The term “aerosol” is used in its conventional sense as referring tovery fine liquid or solid particles carried by a propellant gas underpressure to a site of therapeutic application. A pharmaceutical aerosolof the invention contains a therapeutically active compound, which canbe dissolved, suspended, or emulsified in a mixture of a fluid carrierand a propellant. The aerosol can be in the form of a solution,suspension, emulsion, powder, or semi-solid preparation. Aerosols of theinvention are intended for administration as fine, solid particles or asliquid mists via the respiratory tract of a patient. Various types ofpropellants can be utilized including, but not limited to, hydrocarbonsor other suitable gases. Aerosols of the invention can also be deliveredwith a nebulizer, which generates very fine liquid particles ofsubstantially uniform size within a gas. Preferably, a liquid containingthe active compound is dispersed as droplets, which can be carried by acurrent of air out of the nebulizer and into the respiratory tract ofthe patient.

The term “ameliorating” refers to any therapeutically beneficial resultin the treatment of a disease state, e.g., an influenza-related diseasestate, including prophylaxis, lessening in the severity or progression,remission, or cure thereof.

In general, the phrase “well tolerated” refers to the absence of adversechanges in health status that occur as a result of the treatment andwould affect treatment decisions.

“Synergistic interaction” refers to an interaction in which the combinedeffect of two or more agents is greater than the algebraic sum of theirindividual effects.

The term “in vitro” refers to processes that occur in a living cellgrowing separate from a living organism, e.g., growing in tissueculture.

The term “in vivo” refers to processes that occur in a living organism.

The term “mammal” as used herein includes both humans and non-humans andinclude but is not limited to humans, non-human primates, canines,felines, murines, bovines, equines, and porcines.

The term “sufficient amount” means an amount sufficient to produce adesired effect, e.g., an amount sufficient to modulate proteinaggregation in a cell.

The term “therapeutically effective amount” is an amount that iseffective to ameliorate a symptom of a disease. A therapeuticallyeffective amount can be a “prophylactically effective amount” asprophylaxis can be considered therapy.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of elements is notnecessarily limited to only those elements but can include otherelements not expressly listed or inherent to such process or method.Further, unless expressly stated to the contrary, “or” refers to aninclusive or and not to an exclusive or. For example, a condition A or Bis satisfied by any one of the following: A is true (or present) and Bis false (or not present), A is false (or not present) and B is true (orpresent), and both A and B are true (or present).

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions, or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only,and is not intended to be limiting. As used in this specification andthe appended claims, the singular forms “a”, “an”, and “the” includeplural referents unless the content clearly dictates otherwise. Thus,for example, reference to “a vaccine” includes a combination of two ormore vaccines, and the like.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

Influenza Virus Types

The invention is generally used to treat or prevent influenza virusinfection in mammals There are three types of influenza viruses that canbe targeted by the invention: Influenza A, B, and C. Influenza type Aviruses are divided into subtypes based on two proteins on the surfaceof the virus. These proteins are termed hemagglutinin (H) andneuraminidase (N). Influenza A viruses are divided into subtypes basedon these two proteins. There are 16 different hemagglutinin subtypes H1,H2, H3, H5, H4, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16 and9 different neuraminidase subtypes N1, N2, N3, N4, N5, N6, N7, N8, orN9, all of which have been found among influenza A viruses in wildbirds. Influenza A viruses include A(H1N1) and A(H3N2), both of whichare examples of influenza viruses that can be targeted by the inventionfor treatment or prevention in a mammal Diseases and symptoms typicallycaused by influenza virus infection can include: fever, coughing,sneezing, aches, fatigue, headache, watery eyes, nasal congestion, andabdominal pain. The invention can be used to treat or prevent thesediseases.

Compositions Bordetella Strains

The invention provides a mutated Bordetella strain that can be used asan immunogenic composition or a vaccine to elicit an immune response ina mammal. In one aspect, the mutated Bordetella strain contains amutated ptx gene, a deleted or mutated dnt gene, and a heterologous ampGgene. The heterologous ampG gene product can reduce in large quantitiesthe amount of tracheal cytotoxin that is produced. In one aspect, thestrain is BPZE1. The starting strain which is mutated can be anyBordetella strain including Bordetella pertussis, Bordetellaparapertussis, and Bordetella bronchiseptica. In one aspect the startingstrain used to obtain the mutated Bordetella strain is B. pertussis. Inanother aspect, the strain is a triple mutant Bordetella strain. Inanother aspect, the Bordetella strain is identified by accession numberCNCM 1-3585. In another aspect, the Bordetella strain is identified byaccession number V09/009169.

The invention is not limited to only the mutants described above. Otheradditional mutations can be undertaken such as adenylate cyclase (AC)deficient mutants, lipopolysaccharide (LPS) deficient mutants,filamentous hemagglutinin (FHA), and any of the bvg-regulatedcomponents.

The construction of a mutated Bordetella strain of the invention canbegin with replacing the Bordetella ampG gene in the strain with aheterologous ampG gene. Any heterologous ampG gene known in the art canbe used in the invention. Examples of these can include allgram-negative bacteria that release very small amounts of peptidoglycanfragments into the medium per generation. Examples of gram-negativebacteria include, but are not limited to: Escherichia coli, Salmonella,Enterobacteriaceae, Pseudomonas, Moraxella, Helicobacter,Stenotrophomonas, Legionella, and the like. Typically, by replacing theBordetella ampG gene with a heterologous ampG gene, the amount oftracheal cytoxin (TCT) produced in the resulting strain expresses lessthan 1% residual TCT activity. In another aspect, the amount of TCTtoxin expressed by the resulting strain is between about 0.6% to 1%residual TCT activity or about 0.4% to 3% residual TCT activity or about0.3% to 5% residual TCT activity.

PTX is a major virulence factor responsible for the systemic effects ofB. pertussis infections, as well as one of the major protectiveantigens. Due to its properties, the natural ptx gene can be replaced bya mutated version so that the enzymatically active moiety 51 codes foran enzymatically inactive toxin, but the immunogenic properties of thepertussis toxin are not affected. This can be accomplished by replacingthe arginine (Arg) at position 9 of the sequence with a lysine (Lys)(R9K). Furthermore, a glutamic acid (Glu) at position 129 can bereplaced with a glycine (GIy) (E129G). Generally these amino acidpositions are involved in substrate binding and catalysis, respectively.In other aspects, other mutations can also be made such as thosedescribed in U.S. Pat. No. 6,713,072, incorporated herein by reference,as well as any known or other mutations able to reduce the toxinactivity. In one aspect, allelic exchange can first be used to deletethe ptx operon and then to insert a mutated version.

In another aspect of the invention, the dnt gene can be removed from theBordetella strain using allelic exchange. Besides the total removal, theenzymatic activity can also be inhibited by a point mutation. Since DNTis constituted by a receptor-binding domain in the N-terminal region anda catalytic domain in the C-terminal part, a point mutation in the dntgene to replace Cys-1305 to Ala-1305 inhibits the enzyme activity of DNT(Kashimoto T., Katahira J, Cornejo W R, Masuda M, Fukuoh A, Matsuzawa T,Ohnishi T, Horiguchi Y. (1999) Identification of functional domains ofBordetella dermonecrotizing toxin. Infect. Immun. 67: 3727-32.).

Besides allelic exchange to insert the mutated ptx gene and theinhibited or deleted dnt gene, the open reading frame of a gene can beinterrupted by insertion of a genetic sequence or plasmid. This methodis also contemplated in the invention. Other methods of generatingmutant strains are generally well known in the art.

In one aspect of the invention, the mutated strain is called a BPZE1strain and has been deposited with the Collection Nationale de Culturesde Microorganismes (CNCM) in Paris, France under the Budapest Treaty onMar. 9, 2006 and assigned the number CNCM 1-3585. The mutationsintroduced into BPZE1 generally result in attenuation, but also allowthe bacteria to colonize and persist. Thus, in another aspect, theinvention provides BPZE1, which can induce mucosal immunity and systemicimmunity when administered to a mammal in need thereof. In anotheraspect of the invention, a BPZE1 recombinant strain was constructedwhich expresses three copies of M2e peptide. This strain has beendeposited with the National Measurement Institute (formerly AGAL) inPort Melbourne, Victoria, Australia 3207 under the Budapest Treaty onApr. 27, 2009, and assigned the following accession number V09/009169.M2e is the extracellular portion of the M2 protein from influenza virus.It is highly conserved among all influenza A viruses and has been shownto induce an antibody-mediated protection against influenza A viruses.The recombinant M2e-producing BPZE1 strain can trigger (for example,upon nasal administration of the live bacteria) substantial anti-M2eantibody responses (local and systemic), allowing a significantprotection against H1N1 and H3N2 challenge comparable to the BPZE1bacteria alone.

The mutated Bordetella strains of the invention can be used inimmunogenic compositions for the treatment or prevention of influenzavirus infections. Such immunogenic compositions are useful to raise animmune response, either an antibody response and or a T cell response inmammals. For example, the T cell response can be such that it protects amammal against influenza infection or against itsconsequences/diseases/symptoms.

The mutated Bordetella strains of the invention can be used as livestrains in vaccines or immunogenic compositions. In one aspect, the livestrains are used for nasal administration, while the chemically- or heatkilled strains can be used for systemic or mucosal administration. Inother aspects the stains are attenuated.

In other aspects of the invention, the strains do not include anyheterologous genes other than the heterologous ampG gene describedabove. In yet other aspects, the strains do not include a heterologousexpression platform (See, e.g., WO2007104451). Typically, heterologousexpression platforms carry heterologous antigens. In one aspect, theheterologous expression platform can be used to deliver the heterologousantigens to the respiratory mucosa of a mammal.

Adjuvants

Compositions of the invention can be administered in conjunction withother immunoregulatory agents, including adjuvants. As used herein, theterm “adjuvant” refers to a compound or mixture that enhances an immuneresponse. In particular, compositions can include an adjuvant. Adjuvantsfor use with the invention can include, but are not limited to, one ormore of the following set forth below:

Mineral Containing Adjuvant Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminum salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.,oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates),sulfates, and the like (e.g., see chapters 8 & 9 of Vaccine Design . . .(1995) eds. Powell & Newman ISBN: 030644867X. Plenum.), or mixtures ofdifferent mineral compounds (e.g., a mixture of a phosphate and ahydroxide adjuvant, optionally with an excess of the phosphate), withthe compounds taking any suitable form (e.g., gel, crystalline,amorphous, and the like), and with adsorption to the salt(s) beingpreferred. The mineral containing compositions can also be formulated asa particle of metal salt (WO/0023105).

Aluminum salts can be included in compositions of the invention suchthat the dose of Al₃ ⁺ is between 0.2 and 1.0 mg per dose.

Oil-Emulsion Adjuvants

Oil-emulsion compositions suitable for use as adjuvants in the inventioncan include squalene-water emulsions, such as MF59 (5% Squalene, 0.5%TWEEN® 80, and 0.5% SPAN® 85, formulated into submicron particles usinga microfluidizer). See, e.g., WO90/14837. See also, Podda, “Theadjuvanted influenza vaccines with novel adjuvants: experience with theMF59-adjuvanted vaccine”, Vaccine 19: 2673-2680, 2001.

In other related aspects, adjuvants for use in the compositions aresubmicron oil-in-water emulsions. Examples of submicron oil-in-wateremulsions for use herein include squalene/water emulsions optionallycontaining varying amounts of MTP-PE, such as a submicron oil-in-wateremulsion containing 4-5% w/v squalene, 0.25-1.0% w/v TWEEN® 80(polyoxyelthylenesorbitan monooleate), and/or 0.25-1.0% SPAN® 85(sorbitan trioleate), and, optionally,N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(F-2′-dipalmitoyl-s-n-glycero-3-huydroxyphosphophoryloxy)-ethylamine(MTP-PE), for example, the submicron oil-in-water emulsion known as“MF59” (International Publication No. WO90/14837; U.S. Pat. Nos.6,299,884 and 6,451,325, incorporated herein by reference in theirentirety; and Ott et al., “MF59—Design and Evaluation of a Safe andPotent Adjuvant for Human Vaccines” in Vaccine Design: The Subunit andAdjuvant Approach (Powell, M. F. and Newman, M. J. eds.) Plenum Press,New York, 1995, pp. 277-296). MF59 can contain 4-5% w/v Squalene (e.g.,4.3%), 0.25-0.5% w/v TWEEN® 80, and 0.5% w/v SPAN® 85 and optionallycontains various amounts of MTP-PE, formulated into submicron particlesusing a microfluidizer such as Model 110Y microfluidizer (Microfluidics,Newton, MA). For example, MTP-PE can be present in an amount of about0-500 μg/dose, or 0-250 μg/dose, or 0-100 μg/dose.

Submicron oil-in-water emulsions, methods of making the same andimmunostimulating agents, such as muramyl peptides, for use in thecompositions, are described in detail in International Publication No.WO90/14837 and U.S. Pat. Nos. 6,299,884 and 6,451,325, incorporatedherein by reference in their entirety.

Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA)can also be used as adjuvants in the invention.

Saponin Adjuvant Formulations

Saponin formulations, can also be used as adjuvants in the invention.Saponins are a heterologous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponin from the bark of theQuillaia saponaria Molina tree have been widely studied as adjuvants.Saponin can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations can includepurified formulations, such as QS21, as well as lipid formulations, suchas Immunostimulating Complexs (ISCOMs; see below).

Saponin compositions have been purified using High Performance ThinLayer Chromatography (HPLC) and Reversed Phase High Performance LiquidChromatography (RP-HPLC). Specific purified fractions using thesetechniques have been identified, including QS7, QS17, QS18, QS21, QH-A,QH-B and QH-C. A method of production of QS21 is disclosed in U.S. Pat.No. 5,057,540. Saponin formulations can also comprise a sterol, such ascholesterol (see WO96/33739).

Combinations of saponins and cholesterols can be used to form uniqueparticles called ISCOMs. ISCOMs typically also include a phospholipidsuch as phosphatidylethanolamine or phosphatidylcholine. Any knownsaponin can be used in ISCOMs. For example, an ISCOM can include one ormore of Quil A, QHA and QHC. ISCOMs are further described in EP0109942,WO96/11711, and WO96/33739. Optionally, the ISCOMS can be devoid ofadditional detergent. See WO00/07621.

A description of the development of saponin based adjuvants can be foundat Barr, et al., “ISCOMs and other saponin based adjuvants”, AdvancedDrug Delivery Reviews 32: 247-27, 1998. See also Sjolander, et al.,“Uptake and adjuvant activity of orally delivered saponin and ISCOMvaccines”, Advanced Drug Delivery Reviews 32: 321-338, 1998.

Virosomes and Virus Like Particles (VLPs)

Virosomes and Virus Like Particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins can be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages,QB-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein pl).

Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as:

(1) Non-Toxic Derivatives of Enterobacterial Lipopolysaccharide (LPS)

Such derivatives include Monophosphoryl lipid A (MPL) and 3-O-deacylatedMPL (3 dMPL). 3 dMPL is a mixture of 3 De-O-acylated monophosphoryllipid A with 4, 5 or 6 acylated chains. An example of a “small particle”form of 3 De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689454. Such “small particles” of 3 dMPL are small enough to be sterilefiltered through a 0.22 micron membrane (see EP 0 689 454). Othernon-toxic LPS derivatives include monophosphoryl lipid A mimics, such asaminoalkyl glucosaminide phosphate derivatives e.g., RC-529. See Johnsonet al., Bioorg Med Chem Lett 9: 2273-2278, 1999.

(2) Lipid A Derivatives

Lipid A derivatives can include derivatives of lipid A from Escherichiacoli such as OM-174. OM-174 is described for example in Meraldi et al.,“OM-174, a New Adjuvant with a Potential for Human Use, Induces aProtective Response with Administered with the Synthetic C-TerminalFragment 242-310 from the circumsporozoite protein of Plasmodiumberghei”, Vaccine 21: 2485-2491, 2003; and Pajak, et al., “The AdjuvantOM-174 induces both the migration and maturation of murine dendriticcells in vivo”, Vaccine 21: 836-842, 2003.

(3) Immunostimulatory Oligonucleotides

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention can include nucleotide sequences containing a CpG motif (asequence containing an unmethylated cytosine followed by guanosine andlinked by a phosphate bond). Bacterial double stranded RNA oroligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. Optionally, the guanosine can be replaced with ananalog such as 2′-deoxy-7-deazaguanosine. See Kandimalla, et al.,“Divergent synthetic nucleotide motif recognition pattern: design anddevelopment of potent immunomodulatory oligodeoxyribonucleotide agentswith distinct cytokine induction profiles”, Nucleic Acids Research 31:2393-2400, 2003; WO02/26757 and WO99/62923 for examples of analogsubstitutions. The adjuvant effect of CpG oligonucleotides is furtherdiscussed in Krieg, “CpG motifs: the active ingredient in bacterialextracts?”, Nature Medicine (2003) 9(7): 831-835; McCluskie, et al.,“Parenteral and mucosal prime-boost immunization strategies in mice withhepatitis B surface antigen and CpG DNA”, FEMS Immunology and MedicalMicrobiology (2002) 32:179-185; WO98/40100; U.S. Pat. Nos. 6,207,646;6,239,116 and 6,429,199.

The CpG sequence can be directed to Toll-like receptor (TLR9), such asthe motif GTCGTT or TTCGTT. See Kandimalla, et al., “Toll-like receptor9: modulation of recognition and cytokine induction by novel syntheticCpG DNAs”, Biochemical Society Transactions (2003) 31 (part 3): 654-658.The CpG sequence can be specific for inducing a Th1 immune response,such as a CpG-A ODN, or it can be more specific for inducing a B cellresponse, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed inBlackwell, et al., “CpG-A-Induced Monocyte IFN-gamma-InducibleProtein-10 Production is Regulated by Plasmacytoid Dendritic CellDerived IFN-alpha”, J. Immunol. 170: 4061-4068, 2003; Krieg, “From A toZ on CpG”, TRENDS in Immunology 23: 64-65, 2002, and WOO1/95935.

In some aspects, the CpG oligonucleotide can be constructed so that the5′ end is accessible for receptor recognition. Optionally, two CpGoligonucleotide sequences can be attached at their 3′ ends to form“immunomers”. See, for example, Kandimalla, et al., “Secondarystructures in CpG oligonucleotides affect immunostimulatory activity”,BBRC 306: 948-95, 2003; Kandimalla, et al., “Toll-like receptor 9:modulation of recognition and cytokine induction by novel synthetic GpGDNAs”, Biochemical Society Transactions 31: 664-658, 2003; Bhagat etal., “CpG penta- and hexadeoxyribonucleotides as potent immunomodulatoryagents” BBRC 300: 853-861, 2003, and WO03/035836.

(4) ADP-Ribosylating Toxins and Detoxified Derivatives Thereof.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof canbe used as adjuvants in the invention. For example, the toxin can bederived from E. coli (i.e., E. coli heat labile enterotoxin (LT)),cholera (CT), or pertussis (PTX). The use of detoxified ADP-ribosylatingtoxins as mucosal adjuvants is described in WO95/17211 and as parenteraladjuvants in WO98/42375. In some aspects, the adjuvant can be adetoxified LT mutant such as LT-K63, LT-R72, and LTR192G. The use ofADP-ribosylating toxins and detoxified derivaties thereof, particularlyLT-K63 and LT-R72, as adjuvants can be found in the followingreferences, each of which is specifically incorporated by referenceherein in their entirety: Beignon, et al., “The LTR72 Mutant ofHeat-Labile Enterotoxin of Escherichia coli Enahnces the Ability ofPeptide Antigens to Elicit CD4+T Cells and Secrete Gamma Interferonafter Coapplication onto Bare Skin”, Infection and Immunity 70:3012-3019, 2002; Pizza, et al., “Mucosal vaccines: non toxic derivativesof LT and CT as mucosal adjuvants”, Vaccine 19: 2534-2541, 2001; Pizza,et al., “LTK63 and LTR72, two mucosal adjuvants ready for clinicaltrials” Int. J. Med. Microbiol 290: 455-461, 2003; Scharton-Kersten etal., “Transcutaneous Immunization with Bacterial ADP-RibosylatingExotoxins, Subunits and Unrelated Adjuvants”, Infection and Immunity 68:5306-5313, 2000; Ryan et al., “Mutants of Escherichia coli Heat-LabileToxin Act as Effective Mucosal Adjuvants for Nasal Delivery of anAcellular Pertussis Vaccine: Differential Effects of the Nontoxic ABComplex and Enzyme Activity on Th1 and Th2 Cells” Infection and Immunity67: 6270-6280, 2003; Partidos et al., “Heat-labile enterotoxin ofEscherichia coli and its site-directed mutant LTK63 enhance theproliferative and cytotoxic T-cell responses to intranasallyco-immunized synthetic peptides”, Immunol. Lett. 67: 09-216, 1999;Peppoloni et al., “Mutants of the Escherichia coli heat-labileenterotoxin as safe and strong adjuvants for intranasal delivery ofvaccines”, Vaccines 2: 285-293, 2003; and Pine et al., (2002)“Intranasal immunization with influenza vaccine and a detoxified mutantof heat labile enterotoxin from Escherichia coli (LTK63)” J. ControlRelease 85: 263-270, 2002. Numerical reference for amino acidsubstitutions is preferably based on the alignments of the A and Bsubunits of ADP-ribosylating toxins set forth in Domenighini et al.,Mol. Microbiol 15: 1165-1167, 1995, specifically incorporated herein byreference in its entirety.

Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives can also be used as adjuvants in theinvention. Suitable bioadhesives can include esterified hyaluronic acidmicrospheres (Singh et al., J. Cont. Rele. 70:267-276, 2001) ormucoadhesives such as cross-linked derivatives of poly(acrylic acid),polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides andcarboxymethylcellulose. Chitosan and derivatives thereof can also beused as adjuvants in the invention. See, for example, WO99/27960.

Adjuvant Microparticles

Microparticles can also be used as adjuvants in the invention.Microparticles (i.e., a particle of about 100 nm to about 150 μm indiameter, or 200 nm to about 30 μm in diameter, or about 500 nm to about10 μm in diameter) formed from materials that are biodegradable and/ornon-toxic (e.g., a poly(alpha-hydroxy acid), a polyhydroxybutyric acid,a polyorthoester, a polyanhydride, a polycaprolactone, and the like),with poly(lactide-co-glycolide) are envisioned, optionally treated tohave a negatively-charged surface (e.g., with SDS) or apositively-charged surface (e.g., with a cationic detergent, such asCTAB).

Adjuvant Liposomes

Examples of liposome formulations suitable for use as adjuvants aredescribed in U.S. Pat. Nos. 6,090,406, 5,916,588, and EP 0 626 169.

I. Polyoxyethylene Ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention can also includepolyoxyethylene ethers and polyoxyethylene esters. WO99/52549. Suchformulations can further include polyoxyethylene sorbitan estersurfactants in combination with an octoxynol (WO01/21207) as well aspolyoxyethylene alkyl ethers or ester surfactants in combination with atleast one additional non-ionic surfactant such as an octoxynol(WO01/21152).

In some aspects, polyoxyethylene ethers can include:polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steorylether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether,polyoxyethylene-35-lauryl ether, or polyoxyethylene-23-lauryl ether.

Polyphosphazene (PCPP)

PCPP formulations for use as adjuvants are described, for example, inAndrianov et al., “Preparation of hydrogel microspheres by coacervationof aqueous polyphophazene solutions”, Biomaterials 19: 109-115, 1998,and Payne et al., “Protein Release from Polyphosphazene Matrices”, Adv.Drug. Delivery Review 31: 185-196, 1998.

Muramyl Peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention can include N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-normuramyl-1-alanyl-d-isoglutamine (nor-MDP), andN-acetylmuramyl-1-alanyl-d-isoglutaminyl-1-alanine-2-(1′-2′-dipalmitoyl-s-n-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use as adjuvants inthe invention can include Imiquimod and its homologues, describedfurther in Stanley, “Imiquimod and the imidazoquinolones: mechanism ofaction and therapeutic potential” Clin Exp Dermatol 27: 571-577, 2002and Jones, “Resiquimod 3M”, Curr Opin Investig Drugs 4: 214-218, 2003.

Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioncan include cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4,IL-5, IL-6, IL-7, IL-12, and the like), interferons (e.g.,interferon-gamma), macrophage colony stimulating factor, and tumornecrosis factor.

Adjuvant Combinations

The invention can also comprise combinations of aspects of one or moreof the adjuvants identified above. For example, adjuvant compositionscan include:

-   -   (1) a saponin and an oil-in-water emulsion (WO99/11241);    -   (2) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g., 3        dMPL) (see WO94/00153);    -   (3) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g., 3        dMPL)+a cholesterol;    -   (4) a saponin (e.g., QS21)+3 dMPL+IL-12 (optionally+a sterol)        (WO98/57659);    -   (5) combinations of 3dMPL with, for example, QS21 and/or        oil-in-water emulsions (See European patent applications        0835318, 0735898 and 0761231);    -   (6) SAF, containing 10% Squalane, 0.4% TWEEN® 80, 5%        PLURONIC®-block polymer L121, and thr-MDP, either microfluidized        into a submicron emulsion or vortexed to generate a larger        particle size emulsion.    -   (7) Ribi adjuvant system (RAS), (Ribi Immunochem) containing 2%        Squalene, 0.2% TWEEN® 80, and one or more bacterial cell wall        components from the group consisting of monophosphorylipid A        (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS),        preferably MPL+CWS (Detox); and    -   (8) one or more mineral salts (such as an aluminum salt)+a        non-toxic derivative of LPS (such as 3 dPML).

Aluminum salts and MF59 are examples of adjuvants for use withinjectable influenza vaccines. Bacterial toxins and bioadhesives areexamples of adjuvants for use with mucosally-delivered vaccines, such asnasal vaccines. All adjuvants noted above and others as generally knownin the art to one of ordinary skill can be formulated for intranasaladministration using techniques well known in the art.

Formulations and Carriers

Methods for treatment or prevention of diseases related to influenzavirus infection (described in more detail below) are also encompassed bythe invention. Said methods of the invention include administering atherapeutically effective amount of a composition of the invention. Thecomposition of the invention can be formulated in pharmaceuticalcompositions. These compositions can comprise, in addition to one ormore of the strains, a pharmaceutically acceptable excipient, carrier,buffer, stabilizer, or other materials well known to those skilled inthe art. Such materials should typically be non-toxic and should nottypically interfere with the efficacy of the active ingredient. Theprecise nature of the carrier or other material can depend on the routeof administration, e.g., oral, intravenous, cutaneous or subcutaneous,nasal, intramuscular, or intraperitoneal routes.

Pharmaceutical compositions for oral administration can be in tablet,capsule, powder or liquid form. A tablet can include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil, or synthetic oil. Physiological salinesolution, dextrose, or other saccharide solution or glycols such asethylene glycol, propylene glycol, or polyethylene glycol can beincluded.

For intravenous, cutaneous, or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity, and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,or Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants, and/or other additives can be included, as required.

Administration is preferably in a “therapeutically effective amount” or“prophylactically effective amount” (as the case can be, althoughprophylaxis can be considered therapy), this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofdisease being treated. Prescription of treatment, e.g., decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors, and typically takes account of the disorder to betreated, the condition of the individual patient, the site of delivery,the method of administration and other factors known to practitioners.Examples of the techniques and protocols mentioned above can be found inthe latest edition of Remington's Pharmaceutical Science, MackPublishing Company, Easton, PA (“Remington's”).

Typically, a composition can be administered alone or in combinationwith other treatments, either simultaneously or sequentially dependentupon the condition to be treated.

Methods Administration Routes

Compositions of the invention will generally be administered directly toa mammal Direct delivery can be accomplished by parenteral injection(e.g., subcutaneously, intraperitoneally, intradermal, intravenously,intramuscularly, or to the interstitial space of a tissue), ormucosally, such as by rectal, oral (e.g., tablet, spray), vaginal,topical, transdermal (See e.g., WO99/27961) or transcutaneous (See e.g.,WO02/074244 and WO02/064162), inhalation, intranasal (See e.g.,WO03/028760), ocular, aural, pulmonary or other mucosal administration.Compositions can also be administered topically by direct transfer tothe surface of the skin. Topical administration can be accomplishedwithout utilizing any devices, or by contacting naked skin with thecomposition utilizing a bandage or a bandage-like device (see, e.g.,U.S. Pat. No. 6,348,450).

In some aspects, the mode of administration is parenteral, mucosal, or acombination of mucosal and parenteral immunizations. In other aspects,the mode of administration is parenteral, mucosal, or a combination ofmucosal and parenteral immunizations in a total of 1-2 vaccinations 1-3weeks apart. In related aspects, the route of administration includesbut is not limited to intranasal delivery.

Administration Procedures and Dosages

The invention can include administration of a mutated Bordetella strainto a mammal to elicit an immune response (e.g., a TH1 immune response)capable of impacting an influenza virus, e.g., H3N2. Examples of mutatedBordetella strains of the invention are described above. Typically,administration of the mutated Bordetella strain is used to treat orprevent an influenza virus infection in a mammal, e.g., a human, viaprotective immunity against the influenza virus. In some aspects, themutated Bordetella strain administration is used to prevent influenzainfection by administration prior to the influenza virus infection.Typically, the mutated Bordetella stain is administered to a mammalabout less than 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeksprior to the influenza virus infection.

In one aspect, the method for treating or preventing an infection by aninfluenza virus includes administering to a subject in need thereof asingle dose of a composition of the invention, e.g., BPZE1. In relatedaspects, the administering step is performed mucosally, e.g.,intranasally.

In other aspects, composition of the invention is administered in morethan one dose, e.g., two doses. The number of doses can vary as needed,for example the number of doses administered to a mammal can be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more doses. In one aspect, the method fortreating or preventing an infection by an influenza virus, includesadministering to a subject in need thereof a first immunogeniccomposition of the invention (comprising e.g., BPZE1) followed by asecond immunogenic composition administration (comprising e.g., BPZE1).Typically, the time range between each dose of the composition can beabout 1-6 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 20, 30, 40, 50, 60, 70, 80, 90, or more weeks. In related aspects,the time range between each dose is about 3 weeks. In other aspects,prime-boost-style methods can be employed where a composition of theinvention can be delivered in a “priming” step and, subsequently, acomposition of the invention is delivered in a “boosting” step.

The composition can typically be used to elicit systemic and/or mucosalimmunity, for example to elicit an enhanced systemic and/or mucosalimmunity. For example, the immune response can be characterized by theinduction of a serum IgG and/or intestinal IgA immune response.Typically, the level of protection against influenza infection can bemore than 50%, e.g., 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more. In one aspect, the level of protection can be100%. In other aspects the level of protection is less than 50%, e.g.,20%. In other aspects, the number of bacteria in each dosage is adjustedto attain an effective immune response in a mammal. The number ofbacteria or cfus in each dosage can be about 1, 10, 100, 1000, 10000,100000, 1000000, 5×10⁶, or more or any dosage between said each dosage.

In other aspects the invention can also include co-administration of thecomposition with another agent or agents. Typically, the variouscompositions/agents can be delivered in any order. Thus, in aspectsincluding delivery of multiple different compositions or agents, themutated Bordetella strain need not be all delivered before the agent,e.g., a drug, a siRNA, a miRNA, an immunogenic peptide, or a smallmolecule capable of effecting an influenza infection. Other examples ofagents include neuraminidase inhibitors and M2 inhibitors (adamantanes).For example, the priming step can include delivery of one or more agentsand the boosting can include delivery of one or more mutated Bordetellastrains. In other aspects, multiple administrations of mutatedBordetella strains can be followed by multiple administrations ofagents. Administrations can be performed in any order. Thus, one or moreof the mutated Bordetella strains described herein and one or moreagents can be co-administered in any order and via any administrationroute known in the art, e.g., to elicit an immune reaction.

In the invention, dosage treatment can be according to a single doseschedule or a multiple dose schedule. For example, multiple doses can beused in a primary immunization schedule and/or in a booster immunizationschedule. In a multiple dose schedule, the various doses can be given bythe same or different routes, e.g., a parenteral prime and mucosalboost, a mucosal prime and parenteral boost, and the like. In otheraspects, the dosage regime can enhance the avidity of the antibodyresponse leading to antibodies with a neutralizing characteristic. Anin-vitro neutralization assay can be used to test for neutralizingantibodies (see for example Asanaka et al, J Virology 102: 10327, 2005;Wobus et al., PLOS Biology 2; e432; and Dubekti et al., J MedicalVirology 66: 400).

Tests to Determine the Efficacy or Presence of an Immune Response

One way of assessing efficacy of therapeutic treatment involvesmonitoring infection after administration of a composition of theinvention. One way of assessing efficacy of prophylactic treatmentinvolves monitoring immune responses against the antigens in thecompositions of the invention after administration of the composition.Another way of assessing the immunogenicity of the compositions of theinvention is to isolate the proteins or proteins mixes and to screenpatient sera or mucosal secretions by immunoblot. A positive reactionbetween the protein and the patient serum indicates that the patient haspreviously mounted an immune response to the composition.

Another way of checking efficacy of therapeutic treatment involvesmonitoring infection after administration of the compositions of theinvention. One way of checking efficacy of prophylactic treatmentinvolves monitoring immune responses both systemically (such asmonitoring the level of IgG1 and IgG2a production) and mucosally (suchas monitoring the level of IgA production) against the antigens in thecompositions of the invention after administration of the composition.Typically, serum specific antibody responses are determinedpost-immunization but pre-challenge whereas mucosal specific antibodyresponses are determined post-immunization and post-challenge. Theimmunogenic compositions of the invention can be evaluated in in vitroand in vivo animal models prior to host, e.g., human, administration.

The efficacy of compositions of the invention can also be determined invivo by challenging animal models of infection, e.g., mice, with thecompositions. The compositions can or cannot be derived from the samestrains as the challenge strains. In vivo efficacy models can includebut are not limited to: (i) A murine infection model using humanstrains; (ii) a murine disease model which is a murine model using amouse-adapted strain, such as strains which are particularly virulent inmice; and (iii) a primate model using human isolates.

The immune response induced by the invention can be one or both of a TH1immune response and a TH2 response. The immune response can be animproved or an enhanced or an altered immune response. The immuneresponse can be one or both of a systemic and a mucosal immune response.For example, the immune response can be an enhanced systemic and/ormucosal response. An enhanced systemic and/or mucosal immunity isreflected in an enhanced TH1 and/or TH2 immune response. For example,the enhanced immune response can include an increase in the productionof IgG1 and/or IgG2a and/or IgA. In another aspect the mucosal immuneresponse can be a TH2 immune response. For example, the mucosal immuneresponse can include an increase in the production of IgA.

Typically, activated TH2 cells enhance antibody production and aretherefore of value in responding to extracellular infections. ActivatedTH2 cells can typically secrete one or more of IL-4, IL-5, IL-6, andIL-10. A TH2 immune response can also result in the production of IgG1,IgE, IgA, and/or memory B cells for future protection. In general, a TH2immune response can include one or more of an increase in one or more ofthe cytokines associated with a TH2 immune response (such as IL-4, IL-5,IL-6 and IL-10), or an increase in the production of IgG1, IgE, IgA andmemory B cells. For example, an enhanced TH2 immune response can includean increase in IgG1 production.

A TH1 immune response can include one or more of an increase in CTLs, anincrease in one or more of the cytokines associated with a TH1 immuneresponse (such as IL-2, IFN-gamma, and TNF-alpha), an increase inactivated macrophages, an increase in NK activity, or an increase in theproduction of IgG2a. For example, the enhanced TH1 immune response caninclude an increase in IgG2a production.

Compositions of the invention, in particular, an immunogenic compositioncomprising one or more strains of the invention can be used either aloneor in combination with other agents optionally with an immunoregulatoryagent capable of eliciting a Th1 and/or Th2 response.

The compositions of the invention can elicit both a cell-mediated immuneresponse as well as a humoral immune response to effectively address aninfluenza infection. This immune response will preferably induce longlasting (e.g., neutralizing) antibodies and a cell-mediated immunitythat can quickly respond upon exposure to one or more infectiousantigens in the future.

Subjects and Mammals

Compositions of the invention are typically for preventing or treatinginfluenza virus strains in mammalian subjects, e.g., humans. In someaspects, subjects can include the elderly (e.g., >65 years old),children (e.g., <5 years old), hospitalized patients, healthcareworkers, armed service and military personnel, food handlers, pregnantwomen, the chronically ill, and people traveling abroad. Thecompositions are generally suitable for these groups as well as thegeneral population or as otherwise deemed necessary by a physician.

Kits

The invention also provides kits comprising one or more containers ofcompositions of the invention. Compositions can be in liquid form or canbe lyophilized. Suitable containers for the compositions include, forexample, bottles, vials, syringes, and test tubes. Containers can beformed from a variety of materials, including glass or plastic. Acontainer can have a sterile access port (for example, the container canbe an intravenous solution bag or a vial having a stopper pierceable bya hypodermic injection needle).

The kit can further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution, or dextrose solution. It can also contain othermaterials useful to the end-user, including other pharmaceuticallyacceptable formulating solutions such as buffers, diluents, filters,needles, and syringes or other delivery device(s). The kit can furtherinclude a third component comprising an adjuvant.

The kit can also comprise a package insert containing writteninstructions for methods of inducing immunity, preventing infections, orfor treating infections. The package insert can be an unapproved draftpackage insert or can be a package insert approved by the Food and DrugAdministration (FDA) or other regulatory body.

The invention also provides a delivery device pre-filled with thecompositions of the invention.

The pharmaceutical compositions are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

From the foregoing description, various modifications and changes in thecompositions and methods will occur to those skilled in the art. Allsuch modifications coming within the scope of the appended claims areintended to be included therein. Each recited range includes allcombinations and sub-combinations of ranges, as well as specificnumerals contained therein.

Although the foregoing invention has been described in detail by way ofexample for purposes of clarity of understanding, it will be apparent tothe artisan that certain changes and modifications are comprehended bythe disclosure and can be practiced without undue experimentation withinthe scope of the appended claims, which are presented by way ofillustration not limitation.

Exemplary Aspects

Below are examples of specific aspects for carrying out the invention.The examples are offered for illustrative purposes only, and are notintended to limit the scope of the invention in any way. Efforts havebeen made to ensure accuracy with respect to numbers used (e.g.,amounts, temperatures, and the like), but some experimental error anddeviation should, of course, be allowed for.

The practice of the invention will employ, unless otherwise indicated,conventional methods of protein chemistry, biochemistry, recombinant DNAtechniques and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., T. E.Creighton, Proteins: Structures and Molecular Properties (W.H. Freemanand Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers,Inc., current addition); Sambrook, et al., Molecular Cloning: ALaboratory Manual (2nd Edition, 1989); Methods In Enzymology (S.Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's; Careyand Sundberg Advanced Organic Chemistry 3^(rd) Ed. (Plenum Press)Volumes A and B, 1992).

Materials and Methods Bacterial Strains and Growth Conditions

The bacterial strain used in this study is B. pertussis BPZE1, astreptomycin-resistant Tohamal derivative deleted for the dermonecrotic(DNT)-encoding gene, producing inactivated pertussis toxin (PT) andbackground levels of tracheal cytotoxin (TCT) (22). BPZE1 bacteria weregrown at 37° C. for 72 h on Bordet-Gengou (BG) agar (Difco, Detroit,Mich.) supplemented with 1% glycerol, 10% defibrinated sheep blood and100 μg/ml streptomycin (Sigma Chemical CO., St Louis, Mo.). Liquidcultures were performed as described previously (Menozzi F D, et al.,“Identification and purification of transferring- andlactoferrin-binding proteins of Bordetella pertussis and Bordetellabronchiseptica”, Infect. Immun 59: 3982-3988, 1991) in Stainer-Scholte(SS) medium containing 1 g/l heptakis(2,6-di-o-methyl) β-cyclodextrin(Sigma). Heat-inactivation was performed at 95° C. for 1 hour.

Intranasal Infections. Six to eight week-old female Balb/c mice werekept under specific pathogen-free conditions in Individual VentilatedCages, and all experiments were carried out under the guidelines of theNational University of Singapore animal study board. For BPZE1treatment, sedated mice were intranasally administered once, twice orthree times (as indicated) with approximately 5×106 colony-forming units(cfu) of live or dead BPZE1 bacteria in 20 μl sterile PBS supplementedwith 0.05% TWEEN® 80 (Sigma) (PBST) as previously described (MielcarekN, et al., “Intranasal priming with recombinant Bordetella pertussis forthe induction of a systemic immune response against a heterologousantigen”, Infect immune 65: 544-550, 1997). For influenza infection,sedated mice were nasally administered with approximately 2×10⁶ TCID₅₀of mouse-adapted A/Aichi/2/68 (H3N2) virus passage 10 (Narasaraju T, etal., “Adaptation of human influenza H3N2 virus in a mouse pneumonitismodel: insights into viral virulence, tissue tropism and hostpathogenesis”, Microbes Infect 11: 2-11, 2009) or 4 lethal dose (LD)50of H1N1 (A/PR/8/34) influenza virus (ATCC #VR-95) in 20 μl sterile PBSsupplemented with penicillin and streptomycin. Ten mice per group wereused to determine the survival rates based on body weight loss and themice were euthanized when body weight loss exceeded 20% of the originalbody weight.

Viral Titer Determination

Mouse lungs were harvested and homogenized using mechanical disruption(Omni homogenizer), and tested for the presence of viable virus bytissue culture infectious dose 50 (TCID₅₀) assay using a modified methodreported by the WHO (WHO, “WHO Manual on Animals Influenza Diagnosis andSurveillance” (World Health Organization, Geneva), 2002). Briefly, 90%confluent Madin-Darby Canine Kidney (MDCK) cells in 96-well plates wereinoculated with 100 μl of 10-fold serially diluted lung homogenates.Plates were incubated at 35° C. in a humidified incubator (5% CO₂) for 3days. TCID₅₀ was determined by a reduction in cytopathic effect (CPE) of50%, and the log TCID₅₀/lung was derived. Five mice per group per timepoint were individually assessed.

Histopathologic Examination

Four mice per group were sacrificed and their lungs were harvested 3days post-viral challenge. The lungs were removed and fixed in 10%formalin in PBS. After fixation, the lungs were embedded in paraffin,sectioned, and stained with H&E.

Cellular Infiltrates in Bronchoalveolar Lavage Fluids (BALFs)

Individual BALFs were recovered by injecting 1 ml of sterile PBS intothe lungs of sacrificed animals and performing one lavage step ensuringthat both lungs inflated during the process. BALFs were then centrifugedat 400 g for 10 min, and the supernatant was removed and stored at −80°C. for cytokine detection. Total BALF cell number was determined using ahemocytometer. The cells were spotted onto a glass slide using aCytospin device (Thermo Shandon), and were stained with a modifiedWright staining procedure (Kimman T G, et al., “Development and antigenspecificity of the lymphoproliferation response of pigs to pseudorabiesvirus: dichotomy between secondary B- and T-cell responses”, Immunology86: 372-378, 1995). Identification of the different cell types wasperformed using standard morphological criteria. Results are expressedas the percentage of each cell type in the total cell population. Atotal of 500 cells were considered per slide. Four mice per group wereindividually assessed.

FACS Analysis.

Mice were sacrificed, their lungs were harvested and single cellsuspensions were prepared by digesting the lungs at 37° C. for 15 min in2 ml digestion buffer containing 0.5 mg/ml Liberase (Roche) in RPMI with1% FCS and 2 U/ml DNaseI (Qiagen), followed by centrifugation onFicoll-Paque™PLUS (GE) for 20 min at 600 g and room temperature. Cellswere collected and washed twice with sterile FACS buffer (2% FCS, 5 mMEDTA in PBS). 10⁶ cells were stained with FITC-labeled anti-mouse CD3antibody (eBioscience) and analyzed on CyAn™ ADP cytometer (Dako). Fivemice per group per time point were individually assessed.

Cytokine and Chemokine Analysis

Cytokine and chemokine production in the BALFs supernatants was measuredusing Procarta cytokine profiling kit, according to the manufacturer'sinstructions (Panomics). After incubation with Ab-conjugated beads,detection Abs and streptavidin-PE complexes, samples were run onBio-Plex instrument (Bio-Rad). Levels of the following growth factors,cytokines, and inflammatory mediators were evaluated: GM-CSF, KC, IL-1β,IL-6, IFN-γ and TNF-α, IFN-α, MCP-1 RANTES, IL-10. In addition, TGF-βlevels were measured using a Human/Mouse TGFβ1 ELISA kit (eBioscience)according to the manufacturer's instructions.

Passive Transfer Experiment

A high titer anti-B. pertussis immune serum was generated in 10 adultBalb/c mice nasally infected twice at a 4-week interval with 5×10⁶ cfuof live BPZE1 bacteria. Another group of 10 adult naïve Balb/c mice wereinjected intraperitoneally (ip.) with 10^(5.5) TCID₅₀ ofheat-inactivated human/Aichi/2/68 (H3N2) virus (HI-H3N2) in completeFreund's adjuvant and boosted with the same amount of HI-H3N2 virus inincomplete Freund's adjuvant 2 weeks later. The immune sera from eachmouse group were collected 2 weeks after the boost, pooled and theanti-pertussis and anti-influenza antibody titers were measured byELISA. Moreover, HI-H3N2 serum was tested for the presence ofneutralizing antibodies by neutralization assay. The immune sera werefilter-sterilized, heat inactivated at 56° C. for 30 min and stored at−80° C. until further use. Sera from control naïve mice were alsocollected as negative control.

Six to eight week-old recipient Balb/c mice were ip. injected with 200μl of naïve, anti-BPZE1 or anti-H3N2 immune serum 1 day prior viralchallenge with mouse-adapted H3N2 virus. Body weight losses weremonitored to determine the survival rates. Ten mice per group wereassayed.

T Cell Proliferation Assay

Lymphocyte proliferation was measured by incorporation of tritiated (3H)thymidine as described elsewhere (Bao Z, et al., “Glycogen synthasekinase-3beta inhibition attenuates asthma in mice”, Am J Respir CritCare Med 176: 431-438, 2007). Briefly, spleens from naïve andBPZE1-treated mice (6 mice per group) were collected under asepticcondition and pooled. Single-cell suspensions were prepared andcentrifuged on Ficoll-Paque™PLUS (GE) for 20 min at 600 g at roomtemperature. The isolated splenocytes were seeded in 96-wellround-bottom plates (NUNC) at a density of 2×10⁵ cells/well in 100 μlmedium (RPMI640 supplemented with 10% FCS, 5×10-5 M β-mercaptoethanol, 2mM L-glutamine, 10 mM HEPES, 200 U/ml penicillin, 200 μg/mlstreptomycin). 100 μl medium containing 20 μg/ml of BPZE1 whole celllysate or heat-inactivated 10⁵ TCID₅₀ mouse-adapted H3N2 influenza virus(HI-H3N2) (test antigen) were added to the splenocytes. 100 ul ofnon-infected egg amniotic fluid and 100 ul of medium containing 5 μg/mlconcanavalin A (conA) were used as mock and vitality controls,respectively. After 3 days of incubation at 37° C. in 5% CO₂ atmosphere,the cultures were pulsed with 0.4 μCi [3H]thymidine in 20 μl RPMIcomplete medium. After 18 hrs incubation, cells were harvested, washedand the incorporated radioactivity was measured in TopCount NXT™Microplate Scintillation and Luminescence Counter (PerkinElmer). Resultsare expressed as stimulation index (SI) corresponding to the ratiobetween the mean of [³H]thymidine uptake in the presence of test antigenand the mean of [³H]thymidine uptake in the absence of test antigen. ASI>2 was considered positive. Each sample was assayed in quadruplicate.

IFN-α ELISPOT Assay

The frequency of antigen-specific IFN-γ-producing splenocytes wasdetermined by ELISPOT assay using BD mouse ELISPOT set (BD PharMingen)according to the manufacturer's instructions. Briefly, single-cellsuspensions of individual spleen from naïve and BPZE1-treated mice wereprepared and plated in 96-well microplates (Millipore, Bedford, MA)pre-coated with 100 μl of [5 μg/ml anti-IFN-γ antibody in sterile PBS]overnight at 4° C., washed three times and blocked for 2 hr at roomtemperature with RPMI 1640 containing 10% FCS. Cells were then incubatedwith 20 μg/ml of BPZE1 whole cell lysate or heat-inactivated 10⁵ TCID₅₀mouse-adapted H3N2 influenza virus (HI-H3N2) or 5 μg/ml conA for 12-20hr at 37° C. in 5% CO₂ atmosphere. The plates were then washed followedby addition of biotin-conjugated anti-mouse IFN-γ antibody for 2 hr atroom temperature. After washing, streptavidin-HRP conjugate was addedand incubated at room temperature for 1 hr. Wells were washed again anddeveloped with a 3-amino-9-ethyl-carbazole (AEC) substrate solutionuntil spots were visible. After drying, spot-forming cell numbers werecounted by Bioreader® 4000 (Biosystem). Six animals per group wereindividually assayed.

Statistical Analysis

Unless otherwise stated, bars represent means±SD and averages werecompared using a bidirectional unpaired Student's t test with a 5%significance level with * p<0.05, **p≤0.01 and ***p<0.001.

Example 1: A Single Nasal Administration of Live Attenuated BordetellaPertussis Protects Against H3N2 Influenza Challenge

A mouse-adapted H3N2 influenza virus was obtained through successivelung-to-lung passages of the A/Aichi/2/68 (H3N2) virus into adult Balb/cmice (Narasaraju T, et al., “Adaptation of human influenza H3N2 virus ina mouse pneumonitis model: insights into viral virulence, tissue tropismand host pathogenesis”, Microbes Infect 11: 2-11, 2009). Passage 10(P10) strain exhibited high virulence, causing extra-pulmonary spreadwith necrotic and inflammatory lesions in the various organs of theinfected animals Nasal administration of 2×10⁶ TCID₅₀ of the P10 viralsuspension caused the death of the animals within 4 days (Narasaraju T,et al., “Adaptation of human influenza H3N2 virus in a mouse pneumonitismodel: insights into viral virulence, tissue tropism and hostpathogenesis”, Microbes Infect 11: 2-11, 2009).

Adult Balb/c mice were nasally inoculated with live BPZE1 bacteria andsubsequently challenged either 3 or 6 weeks later with a lethal dose ofmouse-adapted H3N2 virus. Survival rate based on body weight changesindicated that the mice challenged 3 weeks after nasal BPZE1 treatmentwere not significantly protected whereas 60% protection was achievedwhen mice were challenged 6 weeks post-BPZE1 treatment (FIG. 1 ).

Example 2: Live but not Dead Bpze1 Bacteria Protect Against Lethal H3N2Challenge

Adult Balb/c mice were nasally administered once with live or dead BPZE1bacteria and were subsequently challenged with a lethal dose ofmouse-adapted H3N2 virus 6 weeks post-BPZE1 treatment. The resultsshowed that dead bacteria did not provide any significant protectionagainst H3N2 (FIG. 2 ), suggesting that bacterial colonization of themouse lung is necessary to induce the protective mechanisms.

Example 3: Boost Effect

Live BPZE1 bacteria were nasally administered to Balb/c mice twice at a4-week interval prior lethal challenge with mouse-adapted H3N2 virusperformed 4 weeks after the last BPZE1 administration. A 100% protectionrate was obtained for the BPZE1-treated animals with minimal body weightchanges (FIG. 3 ). Similar protection rate was achieved when the viralchallenge was performed 2 weeks after the boost. These data indicatedthat a second nasal administration of live BPZE1 bacteria not onlyenhanced the protection efficacy but also shortened the time necessaryto trigger the protective mechanisms.

Example 4: Bpze1 Bacteria Provide Protection Against H1N1 VirusChallenge

The protective potential of BPZE1 bacteria against influenza A viruseswas further explored. Mice nasally treated once with live BPZE1 bacteriawere not protected against a lethal challenge with human A/PR/8/34(H1N1) influenza A virus performed 6 weeks later (data not shown).However, three consecutive administrations of live BPZE1 bacteriaconferred 50% protection against H1N1 virus (FIG. 4 ). Theseobservations indicated that BPZE1 bacteria can have the potential toprotect against all influenza A viruses although with a variableefficacy.

Example 5: The Viral Load is not Reduced in the Protected Mice

To further characterize the cross-protection against influenza Aviruses, the viral load was quantified in the lungs of mice treatedtwice with BPZE1 bacteria and in control mice. The virus load waschecked 3 days post-infection which corresponds to the peak of virustiter in mice infected with mouse-adapted H3N2 virus (Narasaraju T, etal., “Adaptation of human influenza H3N2 virus in a mouse pneumonitismodel: insights into viral virulence, tissue tropism and hostpathogenesis”, Microbes Infect 11: 2-11, 2009). No significantdifference in the viral load was observed between the two animal groups(FIG. 5 ). This result suggested that the cross-protective mechanismstriggered in the BPZE1-treated animals do not directly target the virusparticles and/or infected cells.

Example 6: Bpze1 Treatment Protects Mice from Influenza-InducedImmunopathology and Lymphocyte Depletion

Lung immunopathology was examined by histology of lung sections frominfected BPZE1-treated animals and control mice. As expected and asdescribed previously (Narasaraju T, et al., “Adaptation of humaninfluenza H3N2 virus in a mouse pneumonitis model: insights into viralvirulence, tissue tropism and host pathogenesis”, Microbes Infect 11:2-11, 2009), the infected control mice displayed severe inflammationwith inflammatory cells, severe bronchopneumonia and interstitialpneumonitis with bronchioles and alveoli filled with necrotic debris aswell as high pulmonary emphysema and moderate edema (FIG. 7A). Incontrast, only mild inflammation, minimal airway and alveolar damage,and mild perivascular/peribronchular damage associated with minimaledema were observed in the lungs of the protected BPZE1-treated mice.

The cell populations present in the broncho-alveolar lavage fluids(BALFs) recovered from protected and non-protected animals were alsoexamined Whereas the total number of cells present in the BALFs fromboth animal groups were comparable (11.8×10⁵ versus 16.1×10⁵), asignificantly higher number of macrophages and lower number ofneutrophils were found in the BPZE1-treated mice (FIG. 6B).

Furthermore, the lymphocyte population present in the lungs of protectedand non-protected mice was analysed. Lymphocyte depletion has indeedbeen reported in mice infected with highly pathogenic H1N1(1918) andH5N1 influenza viruses (Kash J C, et al., “Genomic analysis of increasedhost immune and cell death responses induced by 1918 influenza virus”,Nature 443: 578-581, 2006; Uiprasertkul M, et al., “Apoptosis andPathogenesis of Avian Influenza A (H5N1) Virus in Humans”, Emerg InfectDis 13: 708-712, 2007; Lu X, et al., “A mouse model for the evaluationof pathogenesis and immunity to influenza A (H5N1) viruses isolated fromhumans”, J Virol 73: 5903-5911, 1999) as well as in mice infected withthe mouse-adapted H3N2 virus strain used in this work (Narasaraju T, etal., “Adaptation of human influenza H3N2 virus in a mouse pneumonitismodel: insights into viral virulence, tissue tropism and hostpathogenesis”, Microbes Infect 11: 2-11, 2009). Here, Balb/c mice weretreated twice with BPZE1 bacteria and challenged with mouse-adapted H3N2virus 4 weeks later. The mouse lungs were harvested 3 and 5 dayspost-influenza challenge for FACS analysis of the T cell population.Three days post-viral challenge, the percentage of CD3⁺ T lymphocytes inthe infected control mice and BPZE1-treated mice was comparable to thepercentage found in the animals before challenge (FIG. 6C). However, asignificant CD3⁺ T cell depletion was observed in the infected controlanimals 5 days post-viral challenge (FIG. 6C) as reported before(Narasaraju T, et al., “Adaptation of human influenza H3N2 virus in amouse pneumonitis model: insights into viral virulence, tissue tropismand host pathogenesis”, Microbes Infect 11: 2-11, 2009). In contrast,the T cell population remained constant before and after challenge inthe protected BPZE1-immunized animals (FIG. 6C), suggesting thatBPZE1-treatment prevented influenza-induced lymphocyte depletion.

Example 7: The Production of Pro-Inflammatory Cytokines and Chemokinesis Dampened in the Protected Bpze1-Treated Mice

Major pro-inflammatory cytokines and chemokines were measured in theBALFs recovered from protected BPZE1-treated mice and compared tonon-protected mice, 1 and 3 days after lethal H3N2 virus challenge. Allthe pro-inflammatory cytokines and chemokines measured in the BALFs ofthe protected animal group were significantly lower than those measuredin the non-protected mice (FIG. 7 ). The differences were observed atboth day 1 and day 3 for IL-1β, IL-6, IFN-γ and TNF-α, which are themain pro-inflammatory cytokines contributing to influenza-inducedimmunopathology and correlating with disease severity (de Jong M D, etal., “Fatal outcome of human influenza A (H5N1) is associated with highviral load and hypercytokinemia”, Nat Med 12: 1203-1207, 2006; Beigel JH, et al., “Avian influenza A (H5N1) infection in humans: N Engl J Med353: 1374-1385, 2005; Peiris J S, et al., “Re-emergence of fatal humaninfluenza A subtype H5N1 disease”, Lancet 363: 617-619, 2004; Schmitz N,et al., “Interleukin-1 is responsible for acute lung immunopatholoy butincreases survival of respiratory influenza virus infection”, J Virol79: 6441-6448, 2005). The levels of IFN-α, MCP-1 and RANTES were foundsignificantly reduced 1 day post-viral challenge whereas the productionof IL-12(p70), GM-CSF and KC was lower at day 3 post-challenge.Strikingly, complete suppression of IL-12 production was found in theprotected animal group, consistent with lower levels of IFN-γ.

Furthermore, no significant difference was detected in the levels ofanti-inflammatory cytokines IL-10 and TGF-β between protected andnon-protected animals, ruling out the involvement of type 1 regulatory Tcells (Tr1) in the cross-protection mechanisms (FIG. 7 ).

Example 8: B. Pertussis-Specific Adaptive Immunity is not Involved inthe Cross-Protection

The presence of cross-reactive (and protective) antibodies and/or Tcells in the BPZE1-treated animals was examined Firstly, a BLAST searchfailed to identify any matching epitopes between B. pertussis andinfluenza A H3N2 and H1N1 viruses (data not shown). Secondly, the immuneserum from BPZE1-treated mice did not react with whole H3N2 viralparticles in an ELISA assay, neither did it neutralize the virus in anin vitro neutralization assay (data not shown). Thirdly, a high-titeranti-BPZE1 immune serum did not confer any protection against H3N2lethal challenge in an in vivo passive transfer experiment whereas animmune serum raised against heat-inactivated H3N2 virus gave a 100%protection rate (FIG. 8A). Fourthly, proliferation and IFN-γ ELISPOTassays showed that splenocytes from BPZE1-treated mice did notproliferate and did not produce IFN-γ respectively upon stimulation withH3N2 viral particles (FIGS. 8B & C). Altogether, these data stronglysupport that B. pertussis-specific immunity does not play any role inthe cross-protection against influenza A viruses.

Discussion

Severe respiratory disease and immunopathology together with a highcase-fatality rate have become a hallmark of highly pathogenic avianinfluenza virus infection in humans as well as in other mammalianspecies. However, the underlying mechanisms accounting for the severeimmunopathological effects have yet to be fully elucidated. Inparticular, relationship between viral load, immunopathology and diseaseoutcome remains elusive. Several previous studies have reported areduced mortality rate and immunopathology without significant reductionof the viral burden in animal models of influenza infection; Forexample, reduced inflammatory cell infiltration and pulmonary damage butwith delayed viral clearance was observed in mice with disrupted MIP-1αgene (Cook D N, et al., “Requirement of MIP-1 alpha for an inflammatoryresponse to viral infection”, Science 269: 1583-1585, 1995). Likewise,CCR2 (primary receptor for MCP-1)-deficient mice displayed reducedmortality but a significantly elevated viral burden associated withdecreased pulmonary cell infiltration and tissue damage (Dawson T C, etal., “Contrasting effects of CCR5 and CCR2 deficiency in the pulmonaryinflammatory response to influenza A virus”, Am J Pathol 156: 1951-1959,2000). In contrast, IL-1R knock-out mice showed increased mortalityassociated with delayed viral clearance but less severe pathology(Schmitz N, et al., “Interleukin-1 is responsible for acute lungimmunopatholoy but increases survival of respiratory influenza virusinfection”, J Virol 79: 6441-6448, 2005).

Here, we report that B. pertussis-mediated cross-protection againstinfluenza A viruses does not result a in lower virus load in the lungs.Instead, protected BPZE1-treated mice displayed minimal lungimmunopathology and reduced production of the major pro-inflammatorycytokines and chemokines. Our finding is in agreement with the generalconsensus that disease severity correlates strongly with highcytokines/chemokines levels (La Gruta N L, et al., “A question ofself-preservation immunopathology in influenza virus infection”, ImmunolCell Biol 85: 85-92 2007). Cytokine storm, characterized by excessivelevels of chemokines and cytokines in the serum and lungs due touncontrolled activation of the host's immune system, was indeedcorrelated with the fatal outcome of experimental animals infected withreconstructed 1918 H1N1 and H5N1 influenza viruses (Kash J C, et al.,“Genomic analysis of increased host immune and cell death responsesinduced by 1918 influenza virus”, Nature 443: 578-581, 2006; Simon A K,et al., “Tumor necrosis factor-related apoptosis-inducing ligand in Tcell development: sensitivity of human thymocytes”, Proc Natl Acad SciUSA 98: 5158-5163, 2001; Kobasa D, et al., “Aberrant innate immuneresponse in lethal infection of macaques with the 1918 influenza virus”,Nature 445: 319-323, 2007; Tumpey T M, et al., “Characterization of thereconstructed 1918 Spanish influenza pandemic virus”, Science 310:77-80, 2005) as well as in humans (de Jong M D, et al., “Fatal outcomeof human influenza A (H5N1) is associated with high viral load andhypercytokinemia”, Nat Med 12: 1203-1207, 2006; Beigel J H, et al.,“Avian influenza A (H5N1) infection in humans: N Engl J Med 353:1374-1385, 2005; Uiprasertkul M, et al., “Apoptosis and Pathogenesis ofAvian Influenza A (H5N1) Virus in Humans”, Emerg Infect Dis 13: 708-712,2007; Peiris J S, et al., “Re-emergence of fatal human influenza Asubtype H5N1 disease”, Lancet 363: 617-619, 2004). Furthermore,histological and pathological indicators strongly suggest a key role foran excessive host response in mediating at least some of the extremepathology associated with highly pathogenic influenza viruses. However,the role of each individual cytokine during influenza infection hasremained unclear with often both positive and negative effects; Althoughtheir production can be important for viral clearance through therecruitment and/or activation of immune effector cells on the site ofinfection, their inflammatory properties can also lead to tissue damage(La Gruta N L, et al., “A question of self-preservation immunopathologyin influenza virus infection”, Immunol Cell Biol 85: 85-92 2007).

The reduced production of pro-inflammatory cytokines and chemokines inthe respiratory tract of the protected BPZE1-treated animals likelyimpacted on cellular infiltration and immune cell activation; Asignificantly lower neutrophil counts was indeed observed in the BALFsof the protected animals consistent with lower levels of KC and TNF-α,both cytokines being involved in the recruitment and activation ofneutrophils in the infected tissues (La Gruta N L, et al., “A questionof self-preservation: immunopathology in influenza virus infection”,Immunol Cell Biol 85: 85-92 2007; Kips J C, et al., “Tumor necrosisfactor causes bronchial hyperresponsiveness in rats”, Am Rev Respir Dis145: 332-336, 1992; Headley A S, et al., “Infections and theinflammatory response in acute respiratory distress syndrome”, Chest111: 1306-1321, 1997). Furthermore, the suppressed production of IL-12measured in the protected animals likely impaired the activation of someimmune cells such as natural killer (NK) cells and cytotoxic CD8⁺ Tcells. Both cell types have been described potentially harmful andinvolved in immunopathology upon release of inflammatory mediators (LaGruta N L, et al., “A question of self-preservation: immunopathology ininfluenza virus infection”, Immunol Cell Biol 85: 85-92 2007).Consistently, IFN-γ production, a hallmark for NK and CD8⁺ T cellactivation, was found significantly lower in the BALFs recovered fromthe protected animals. In addition, the lower IFN-γ production canaffect the neutrophil responses to the virus, including oxidative burst,induction of antigen presentation and chemokine production (Ellis T Nand Beaman B L, “Interferon-gamma activation of polymorphonuclearneutrophil function”, Immunology 112: 2-11, 2004; Farrar M A andSchreiber R D, “The molecular cell biology of interferon-gamma and itsreceptor”, Annu Rev Immunol 11: 571, 1993).

Interestingly, a significantly higher number of macrophages wereobserved in the BALFs of protected BPZE1-treated mice despite lowerlevels of MCP-1 and GM-CSF, involved in monocytes recruitment anddifferentiation into macrophages, respectively. However, note should betaken that alveolar macrophages (AM) and not tissue resident macrophagesconstitute the main macrophage population recovered in the BALFs(Jakubzick C, et al., “Modulation of dendritic cells trafficking to andfrom the airways”, J Immunol 176: 3578-3584, 2006). It is thusconceivable that the protected mice can display a reduced macrophagepopulation in their lung tissues compared to infected control mice.

AM have been shown to display suppressive effects on the inflammationreaction by regulating T-cell function (Strickland D H, et al.,“Regulation of T-cell function in lung tissue by pulmonary alveolarmacrophages”, Immunology 80: 266-272, 1993), and by suppressingdendritic cells maturation (Holt P G, et al., “Down-regulation of theantigen presenting function(s) of pulmonary dendritic cells in vivo byresident alveolar macrophages”, J Exp Med 177: 397-407, 1993; Bilyk Nand Holt P G, “Inhibition of the immunosuppressive activity of residentpulmonary alveolar macrophages by granulocyte/macrophage colonystimulating factor”, J Exp Med 177: 1773-1777, 1993; Stumbles P A, etal., “Airway dendritic cells: co-ordinators of immunological homeostasisand immunity in the respiratory tract”, APMIS 111: 741-755, 2003) andmigration to the mesenteric lymph nodes (Jakubzick C, et al.,“Modulation of dendritic cells trafficking to and from the airways”, JImmunol 176: 3578-3584, 2006). One can thus hypothesize that the highernumber of alveolar macrophages induced upon influenza challengecontributed to the suppression/control of the inflammation in theprotected BPZE1-treated animals.

Furthermore, we found that the CD3⁺ T cell population remained unchangedin the protected BPZE1-treated animals upon viral challenge whereas asignificant reduction in the proportion of CD3⁺ T cells was found in theinfected control mice. Lymphocyte depletion during highly pathogenicinfluenza infection has been previously reported (Kash J C, et al.,“Genomic analysis of increased host immune and cell death responsesinduced by 1918 influenza virus”, Nature 443: 578-581, 2006;Uiprasertkul M, et al., “Apoptosis and Pathogenesis of Avian Influenza A(H5N1) Virus in Humans”, Emerg Infect Dis 13: 708-712, 2007; NarasarajuT, et al., “Adaptation of human influenza H3N2 virus in a mousepneumonitis model: insights into viral virulence, tissue tropism andhost pathogenesis”, Microbes Infect 11: 2-11, 2009; Lu X, et al., “Amouse model for the evaluation of pathogenesis and immunity to influenzaA (H5N1) viruses isolated from humans”, J Virol 73: 5903-5911, 1999) andexperimental evidences have suggested apoptosis as a potential mechanism(Uiprasertkul M, et al., “Apoptosis and Pathogenesis of Avian InfluenzaA (H5N1) Virus in Humans”, Emerg Infect Dis 13: 708-712, 2007; Lu X, etal., “A mouse model for the evaluation of pathogenesis and immunity toinfluenza A (H5N1) viruses isolated from humans”, J Virol 73: 5903-5911,1999). Since no difference in the viral load was observed betweenprotected and non-protected animals, lymphocyte apoptosis can thus notbe a direct cytolytic effect of the virus itself. Instead, our data areconsistent with previous studies which suggested that in H5N1-infectedhumans and mice, lymphocyte apoptosis was likely caused by cytokinedysregulation and overactivation of the host immune response(Uiprasertkul M, et al., “Apoptosis and Pathogenesis of Avian InfluenzaA (H5N1) Virus in Humans”, Emerg Infect Dis 13: 708-712, 2007; Maines TR, et al., “Pathogenesis of emerging avian influenza viruses in mammalsand the host innate immune response”, Immunol Rev 225: 68-84, 2008). Inparticular, TNF-α and related TNF-superfamily members includingTNF-related apoptosis inducing ligand (TRAIL) are known to induce T cellapoptosis (Simon A K, et al., “Tumor necrosis factor-relatedapoptosis-inducing ligand in T cell development: sensitivity of humanthymocytes”, Proc Natl Acad Sci USA 98: 5158-5163, 2001; Wang J, et al.,“The critical role of LIGHT, a TNF family member, in T celldevelopment”, J Immunol 167: 5099-5105, 2001). Consistently, lowerlevels of TNF-α were measured in the BALFs of protected BPZE1-treatedmice upon influenza challenge, thus potentially translating into lower Tcell apoptosis.

The protective mechanisms responsible for the cross-protection have yetto be identified. However, our data demonstrate that B.pertussis-specific adaptive immunity (including cross-reactiveantibodies and T cells) is not involved. The observation that live butnot dead BPZE1 bacteria confer protection, indicate that bacterial lungcolonization, i.e. a prolonged exposure to the host immune system, isnecessary to induce the protective mechanism(s). Moreover, induction ofthe protective mechanisms necessitates more than 3 weeks to develop asmice treated once with live BPZE1 bacteria and challenged 3 weeks laterwith influenza H3N2 virus were not significantly protected. However, asecond BPZE1 treatment allowed to shorten the time necessary to inducethe protective mechanisms and enhance the protection rate, suggestingthat some memory cells have been produced upon priming that are capableof responding faster and to a greater extent upon a second encounterwith BPZE1 bacteria. Finally, it was observed that three consecutivenasal administrations of live BPZE1 bacteria were necessary to confer50% protection against human A/PR/8/34 (H1N1) influenza virus. Thedifferential protection rates achieved upon H3N2 and H1N1 viruschallenge suggest that the molecular disease-mechanisms induced by bothviruses are different. However, even though the protection efficiencycan vary between subtypes, B. pertussis remains a promising universalvaccine against influenza A viruses.

Much of the activity of B. pertussis virulence factors is dedicated toimmunomodulation so as to suppress, subvert and evade the host defensesystem (Carbonetti N H, “Immunomodulation in the pathogenesis ofBordetella pertussis infection and disease”, Curr Opin Pharmacol 7: 1-7,2007). The immune response to B. pertussis is initiated and controlledthrough toll-like receptor (TLR)-4 signaling, inducing theanti-inflammatory cytokine IL-10 production by dendritic cells (DCs)which could inhibit inflammatory responses and limit pathology in theairways (Higgins S C, et al., “Toll-like receptor 4-mediated innateIL-10 activates antigen-specific regulatory T cells and confersresistance to Bordetella pertussis by inhibiting inflammatorypathology”, J Immunol 171: 3119-3127, 2003). Filamentous hemagglutinin(FHA), the major adhesin produced in B. pertussis, was shown tostimulate IL-10 production and inhibit TLR-induced IL-12 production bymacrophages and DCs, resulting in the development of IL-10 secretingtype 1 T-regulatory (Tr1) cells (McGuirk P, et al., “Pathogen-specific Tregulatory 1 cells induced in the respiratory tract by a bacterialmolecule that stimulates interleukin 10 production by dendritic cells: anovel strategy for evasion of protective T helper type 1 responses byBordetella pertussis”, J Exp Med 195: 221-231, 2002). Interestingly, thesystemic administration of FHA was recently found to reduce intestinalinflammation in a T-cell mediated model of colitis, supporting theanti-inflammatory role of FHA (Braat H, et al., “Prevention ofexperimental colitis by parenteral administration of a pathogen-derivedimmunomodulatory molecule”, Gut 56: 351-357, 2007). However, here nodifference in the production of IL-10 and TGF-□ was found betweenprotected and non-protected mice, ruling out a potential involvement ofFHA-mediated induction of Tr1 in the cross-protection against influenzaA viruses conferred by B. pertussis.

We report here that in a Balb/c mouse model of severe pneumonitis, priornasal administration of an attenuated strain of Bordetella pertussis(BPZE1), the etiologic agent of whooping cough, provided effective andsustained protection against lethal challenge with a mouse-adapted H3N2influenza A virus and to a lesser extent against human H1N1 (A/PR/8/34)influenza A virus. Although the cellular and molecular players involvedin this cross-protection have yet to be identified, our data indicatethat B. pertussis-specific adaptive immunity and Tr1-mediateddown-regulation are likely not involved. Importantly, we found that thecross-protection does not result in viral load reduction. Instead,protected BPZE1-treated mice displayed minimal lung immunopathology,consistent with reduced neutrophil infiltration and lower production ofa variety of major pro-inflammatory cytokines and chemokines in theirBALFs. Our findings thus strongly suggest that protection againstinfluenza A viruses induced-lethal pneumonitis can be achieved throughattenuation of inflammation and dampening of the cytokine storm, anddemonstrate the potential use of BPZE1 bacteria as an effectiveprophylactic means of protecting against virulent influenza A virusinfections.

All publications and patent applications cited in this specification areherein incorporated by reference in their entirety for all purposes asif each individual publication or patent application were specificallyand individually indicated to be incorporated by reference for allpurposes.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications can be made thereto without departing from the spiritor scope of the appended claims.

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What is claimed is:
 1. A method of reducing lung immunopathology in amammalian subject infected with an influenza type A virus, the methodcomprising the step of: prior to the subject becoming infected with theinfluenza type A virus, administering to the subject a sufficient amountof a live attenuated, mutated Bordetella pertussis strain to elicit animmune response which reduces influenza type A-induced lungimmunopathology in the subject, wherein the live attenuated Bordetellapertussis strain comprises a mutated pertussis toxin (ptx) gene.
 2. Themethod of claim 1, wherein the immune response elicited is sufficient toconfer at least a 50% level of protection.
 3. The method of claim 1,wherein the strain is administered intranasally.
 4. The method of claim1, wherein at least a first dose and a second dose of the strain areadministered to the subject.
 5. The method of claim 4, wherein thesecond dose is administered to the subject one to three weeks from theadministration of the first dose.
 6. The method of claim 1, wherein thelive attenuated Bordetella pertussis strain comprises a heterologousampG gene.
 7. The method of claim 1, wherein the live attenuatedBordetella pertussis strain comprises a deleted or mutated dermonecrotic(dnt) gene.
 8. The method of claim 6, wherein the live attenuatedBordetella pertussis strain comprises a deleted or mutated dermonecrotic(dnt) gene.